This proposal is aimed at developing tools and techniques to image single cells at high resolution and reveals the role of epigenetic machinery in chromatin architecture regulation in vivo. Epigenetically regulated changes in chromatin organization are a fundamental component of gene regulation. While a multitude of protein players and epigenetic marks are known, how each of these factors affects chromatin structure is not fully understood, particularly in the context of development. This proposal will examine two epigenetically regulated systems of mammalian chromatin organization, olfactory receptor gene choice and X chromosome inactivation, utilizing a novel, high resolution imaging technique, called soft X-Ray tomography (SXT). SXT allows examination of intact, unfixed and unstained cells to a resolution level similar to electron microscopy. This powerful imaging system, which captures native chromatin architecture, will be employed in combination with innovative labeling approaches and sophisticated genetic and epigenetic manipulations to reveal the molecular underpinnings of higher order chromatin architecture and the 3-dimensional organization of the mammalian nucleus. Because both olfactory gene choice and X-inactivation are examples of developmentally regulated epigenetic gene regulation that are experimentally tractable, this approach will provide significant understanding to the regulatory logic of these two essential processes.